Epidermal and dermal equivalents

ABSTRACT

The present invention relates to the treatment of skin defects by organotypically-cultured autologous keratinocytes isolated from the outer root sheath of anagen or growing hair. Methods for primary, as well as subsequent organotypic cultures (i.e., epidermal equivalents) in fully-defined media supplemented by autologous human serum and substances isolated form blood components, with minimal allogeneic biological supplements, are disclosed herein. Techniques to prepare epidermal equivalents for transplantation by use of a biocompatible glue are also disclosed herein.

FIELD OF THE INVENTION

The invention relates to the field of cell culture of human keratinocyteprecursor and dermal fibroblast cells. The invention also relates to theuse of cultured keratinocyte precursor cells in the repair of skindefects by skin grafting procedures.

BACKGROUND OF THE INVENTION

The healing of skin defects progresses through three general phases; (i)inflammation, (ii) wound cell migration and mitosis, and (iii)extracellular matrix production and remodeling. The ordered sequence ofthese events is thought to be orchestrated by interactions among cells,growth factors, and extracellular matrix proteins. A crucial step ofskin wound healing is epidermal regeneration (i.e.,re-epithelialization). Besides interfollicular epidermal keratinocytesfrom the wound edges, the outer root sheath (ORS) cells from residualhair follicles also contribute to this process (see e.g., Eisen et al.,15 J. Invest. Dermatol. 145-155 (1955)). The ORS of hair follicles iscomprised largely of undifferentiated keratinocytes that encompass thecylindrical structures of the hardened inner root sheath and the hairshaft (see e.g., Montagna & Parakkal, In: The Structure and Function ofSkin 172-258 (Academic Press New York, N.Y., 1974)). Recent literaturehas also indicated that ORS cells are at a lower level of commitment todifferentiation than the basal interfollicular keratinocytes (see e.g.,Coulombe et al., 109 J. Cell Biol. 2295-2312 (1989); Limat et al., 194Exp. Cell Res. 218-227 (1991); Limat et al., 275 Cell Tissue Res.169-176 (1994)), and label-retaining cells have been detected in theanimal as well as the human ORS region near the bulge area whichpossibly represent stem cells for skin epithelial tissues (see e.g.,Cotsarelis et al., 61 Cell 1329-1337 (1990); Kobayashi et al., 90 Proc.Nat. Acad. Sci. USA 7391-7395 (1993); Yang et al., 105 J. Invest.Dermatol. 14-21 (1993); Rochat et al., 76 Cell 1073-1076 (1994); Moll,105 J. Invest. Dermatol. 14-21 (1995)). Additionally, human ORS cellswhich are isolated from plucked anagen scalp hair follicles can beexpanded extensively in vitro (see e.g., Weterings et al., 104 Brit. J.Dermatol. 1-5 (1981); Limat & Noser, 87 J. Invest. Dermatol. 485-488(1986); Imcke et al., 17 J. Am. Acad. Dermatol. 779-786 (1987); Limat etal., 92 J. Invest. Dermatol. 758-762 (1989)). Under conventionalsubmerged culture conditions, ORS cells resemble interfollicularepidermal keratinocytes by both morphologic and biochemical (e.g.,keratin profiles) criteria (see e.g., Stark et al., 35 Differentiation236-248 (1987); Limat et al., 92 J. Invest. Dermatol. 758-762 (1989);Lirnat et al., 642 Ann. N.Y. Acad. Sci. 125-147 (1991)). In organotypicco-cultures with human dermal fibroblasts (i.e., under conditionsmimicking the epidermal environment), ORS cells with respect tohistological, immunohistological, ultrastructural and biochemicalcriteria develop a stratified epithelium reminiscent of regeneratingepidermis (see e.g., Lenoir et al., 130 Dev. Biol. 610-620 (1988); Limatet al., 194 Exp. Cell Res. 218-227 (1991); Limat et al, 642 Ann. N.Y.Acad. Sci. 125-147 (1991)). If such organotypic cultures are graftedonto nude mice, ORS cells form a regular neo-epidermis that is underhomeostatic control (see e.g., Limat et al., 59 Transplantation1032-1038 (1995)). Thus, human ORS cells are of considerable interestfor clinical application.

In the previous decade, interest has focused on the use of culturedepithelial cells for wound coverage. First, sheets of culturedautologous interfollicular keratinocytes were grafted successfully onacute wounds, mainly in the treatment of larger third degree burns (seee.g., O'Connor et al, 1 Lancet 75-78 (1981); Compton et al. 60 Lab.Invest. 600-612 (1989)) but also of epidermolysis bullosa (see e.g.,Carter et al. 17 J. Am. Acad. Dermatol. 246-250 (1987)), pyodermagangrenosum (see e.g., Dean et al, 26 Ann. Plast. Surg. 194-195 (1991);Limova & Mauro, 20 J. Dermatol. Surg. Oncol. 833-836 (1994)), and woundsafter excision of giant congenital nevi (see e.g., Gallico et al., 84 J.Plast. Reconstr. Surg. 1-9 (1989)) or separation of conjoined twins (seee.g., Higgins et al., 87 J. Royal Soc. Med. 108-109 (1994)).

In contrast to the treatment of such acute wounds, the grafting ofchronic wounds (e.g. leg ulcers) with cultured keratinocytes has beenmuch less successful. Allografts do not result in a permanent “take”(see e.g., Fabre. 29 Immunol. Lett. 161-166 (1991)) and thus may beclassified as a “quite effective but expensive biological dressing” (seePhillips et al., 21 J. Am. Acad. Dermatol. 191-199 (1989). Areproducible, major definite “take” of autologous keratinocyrte graftedby various modalities including: sheets of submerged keratinocytecultures consisting of only a few, noncornified cell layers (Hetton etal, 14 J. Am. Acad. Dermatol. 399-405 (1986); Leigh & Purkis, 11 Clin.Exp. Dennatol. 650-652 (1986); Leigh et al, 117 Brit. J. Dermatol.591-597 (1987); Harris et al., 18 Clin. Exp. Dermatol. 417-420 (1993)),trypsinized single cells attached to collagen-coated dressings (Brysk etal., 25 J. Am. Acad. Dermatol. 238-244 (1991)), skin equivalents (Mol etal., 24 J. Am. Acad. Dennatol. 77-82 (1991)) has yet to be convincinglydocumented within the scientific literature. The same lack ofquantitative findings also holds true for various reports on thegrafting of freshly isolated, autologous interfollicular keratinocytes(Hunyadi et al., 14 J. Dermatol. Surg. Oncol. 75-78 (1988)) or ORS cells(Moll et al. 46 Hautarzt 548-552 (1995)) fixed to the wound bed by theuse of a fibrin glue. However, it should be noted that the disadvantagesof the bovine serum used during cultivation of the keratinocytes maycontribute to reduced “take” rate, due to the fact that it resists inkeratinocytes (see e.g., Johnson et al., 11 J. Burn Care Rehab. 504-509(1990)).

SUMMARY OF THE INVENTION

Prior to the disclosure of the present invention herein, the standardmethodology for the generation of a primary culture of ORS keratinocytesconsisted of the plucking of an anagen (i.e., growing hair shaft) hairfollowed by a careful microscopic dissection to remove the hair bulbsand the infundibular hair shaft. The resulting outer root sheath wasthen placed on the culture insert for initiation of the primarykeratinocyte culture. However, numerous subsequent studies(approximately 200), wherein the anagen hair was placed directly on theculture insert without performing the initial micro-dissection to removethe hair bulbs and the infundibular hair shaft, have demonstrated thatsuch tedious and time-consuming dissection of the plucked anagen hairwas not required. This has served to markedly simplify the handlingprocess, reduce the risk for contamination, and resulted in moreefficient initiation of keratinocyte cell plating.

Accordingly, it is an object of the present invention to provideimproved and simplified methods for the generation of keratinocytes orkeratinocyte precursors from outer root sheath cells (ORS cells) infully defined culture conditions for the treatment of various types ofskin defects (e.g., chronic wounds such as leg ulcers, diabetic ulcers,pressure sores, and the like) in both humans and animals. In addition totheir use in the treatment of wounds, keratinocytes may also be used inplastic and cosmetic surgery, or whenever there is a demand for suchskin support (e.g., post operative following the removal of tattoos,naevi, skin cancer, papillomas, after amputation, in sex transformationor re-virgination, rejuvenation of actinically damaged skin after skinresurfacing, tympanoplasty, epithelialization of external ear canal, andthe like).

These aforementioned objectives are accomplished by explantation andculture of plucked, anagen or growing hairs in toto upon microporousmembranes carrying human fibroblast feeder cells at their under-surface.In such primary cultures, large numbers of ORS cells can be easily andrepeatedly obtained, irrespective of the donor's chronological age. SuchORS cells may be used for the subsequent preparation of complex skin,i.e., dermo-epidermal, or epidermal equivalents or kept frozen andstored in order to use them at a later time point.

The subsequent preparation of skin or epidermal equivalents is achievedby the “seeding” of these ORS cells upon a modified, microporousmembrane carrying fibroblast feeder cells (most preferablygrowth-arrested/limited human dermal fibroblast “feeder cells”) at theirunder-surface. During culture, these ORS cells undergo tissuedifferentiation which has been demonstrated to be similar to that ofnormal epidermis. This finding is most probably due to a largecompartment of proliferating cells. The modified culture conditionswhich are disclosed herein are important for the successful treatment ofchronic wounds with epidermal equivalents generated in vitro fromautologous ORS cells.

A further object of the present invention is to provide improved culturesystems for ORS-derived keratinocytes by adhering the anagenic hair ontoa polymeric microporous membrane coated with one or more molecules ofextracellular matrix origin. These improved cultures of ORS cells,designated as skin equivalents or epidermal equivalents, may be used totreat skin defects, especially chronic wounds.

Yet another object of the present invention is to produce skin orepidermal equivalents using a reduced concentration of allogenic orhomologous serum. This greatly mitigates the risk of diseasetransmission, for example, by clinical use of blood products, by the useof autologous or homologous human serum and substances derived orreleased from blood components (e.g., blood platelets) for supplementsin in vitro culturing steps.

A further object of the present invention is a methodology which reducesthe probability of mechanical damage (e.g., separation of the variousconstituent layers) of the skin or epidermal equivalents duringtransport prior to transplantation.

The clinical advantages of the methodology of the present invention, ascompared to grafting techniques of chronic wounds which have beenpreviously utilized, include, but are not limited to: noninvasiveness(so that the cells are available repeatedly), the lack of need forsurgical facilities or anesthesia during the grafting procedure, and ashort immobilization period of only 2 hours required following thegrafting procedure.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice of the present invention, the preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein in their entirety by reference.

The term “keratinocyte layer” as used herein means an in vitro generatedkeratinocyte tissue culture with more or less differentiated structure.The term “epidermal equivalent” as used herein means an in vitrogenerated organotypic tissue culture resembling in its histologicalstructure the natural epidermis especially concerning the stratificationand development of the horny layer. A normal stratified epidermisconsists of a basal layer of small cuboidal cells, several spinouslayers of progressively flattened cells, a prominent granular layer andan orthokeratotic horny layer. All these layers can be detected in theepidermal equivalents that are subject of the invention. Localization ofthose epidermal differentiation products that have been assayed byimmunohistochemistry (e.g. keratins, involucrin, filaggrin, integrins)is similar to that found in normal epidermis.

The term “autologous” as used herein means: (i) that biological materialto be transplanted is derived from the individual to be treated withepidermal equivalents; or (ii) that biological material added to tissuecultures comes from the donor of the cells for tissue culture.

The term “homologous” as used herein means: (i) that biological materialto be transplanted is derived from one or more individuals of the samespecies as the individual to be treated with epidermal equivalents; or(ii) that biological material added to tissue cultures comes from one ormore individuals of the same species as the donor of cells for thetissue culture.

The term “organotypic culture” and the like, refers to culture of cellsunder conditions that promote differentiation of the cells. Underconditions of organotypic culture, proliferation of the cells is slowedcompared to culture under “proliferative” conditions such as primaryculture conditions, and may be completely stopped. In the present case,an important condition for organotypic culture is maintenance of thecells at the air-liquid interface, a so-called “lifted” culturingcondition.

The term “releasate from blood components” (e.g., blood platelets) asused herein means any combination of cytokines or other growth factorsobtained from blood components (e.g., blood platelets). Plateletsstimulated with, for example, thrombin release the content of theiralpha granules into the surrounding medium. Alpha granules usuallycontain several cytokines (e.g., platelet derived growth factor (PDGF),epidermal growth factor (EGF), transforming growth factors alpha andbeta (TGF alpha/beta), platelet factor 4 (PF-4), platelet basic protein(PBP)). However, it is possible to obtain cytokines and other growthfactors from platelets by other methods than stimulating with thrombin.Moreover, other blood components produce growth factors and cytokines aswell. Monocytes, for example, produce IL-1, TNF alpha. IL-6 and othersubstances of interest.

General Method for Preparing Epidermal Equivalents from ORS Cells.Keratinocyte precursor cells are selected from outer root sheath (ORS)of anagen or growing hair which is derived from the individual which isto be subsequently treated with epidermal equivalents. In general,approximately 40 hair follicles are plucked from the scalp, and those inthe anagen phase (i.e., a growing hair shaft) are then selected underthe dissecting microscope. A total of four weeks of culture is usuallyrequired in order to obtain approximately 1 cm² of epidermal equivalentsfrom five hair follicles. However, with improved culture andfermentation techniques it may be possible to get a higher yield (i.e.,a larger area of epidermal equivalents, within this period of time).

The previous standard method for the generation of a primary culture ofORS keratinocytes consisted of the plucking of an anagen (i.e., growinghair shaft) hair followed by a careful microscopic dissection to removethe hair bulbs and the infundibular hair shaft. The resulting outer rootsheath (ORS) was then placed on the culture insert for initiation of theprimary keratinocyte culture. However, numerous subsequent studies(approximately 200), wherein the anagen hair was placed directly on theculture insert without performing the initial micro-dissection to removethe hair bulbs and the infundibular hair shaft, have demonstrated thatsuch tedious and time-consuming dissection of the plucked anagen hairwas not required. This has served to markedly simplify the handlingprocess, reduce the risk for contamination, and resulted in moreefficient initiation of keratinocyte cell plating.

The selected anagen hairs were incubated in an appropriate rinsingbuffer containing various anti-microbial and anti-fungal agents (e.g.,fungizone, penicillin, and streptomycin). Following this procedure, theentire plucked anagen hair is placed directly on the culture insert andallowed to grow for several days, preferably 7-14, days, and morepreferably 8 to 10 days. An optional, additional step is comprised ofpassaging the primary culture and performing a secondary culture inorder to obtain more cellular material for the preparation of largerareas of epidermal equivalents.

The culture insert, a microporous membrane coated with one or moreextracellular matrix substances (e.g., fibrin, fibronectin, collagens,laminins or hyaluronan or mixtures thereof), carries agrowth-arrested/limited feeder cell system on its undersurface. Thecoating of the membrane insert with such extracellular matrix substancesprovides for: (i) an enhanced culture surface for the initial attachmentof the anagen hair (i.e., it sticks easily and remains stationary); (ii)a surface which significantly enhances the migration of the ORSkeratinocytes away from the outer root sheath (ORS) anagen hairfollicles- and (iii) increased growth rates of the spreading ORSkeratinocytes (i.e., the overall culture time needed for production offully differentiated skin or epidermal equivalents) can be reduced tothree weeks, instead of four.

The aforementioned growth-arrested/limited feeder cell system located onthe under surface of the microporous insert membrane is comprised ofprimary dermal fibroblasts obtained from a human skin biopsy. Theprimary dermal fibroblasts are treated with mitomycin-C for 4 to 6 hoursprior to their use as a “feeder cell layer” for the plucked anagen hairand then plated on the underside of the culture insert. Growtharrest/limitation is induced by either mitomycin-C or X-ray treatmentor, preferably, the reduced serum concentration below 5%, and preferably2%. It should be noted that, although some cultures had been performedusing 10% fetal calf serum (FCS; Boehringer Mannheim, Germany), thecurrent utilization of human serum, in order to reduce the number ofallogeneic ingredients, was found to provide markedly superior outgrowthand proliferation of the ORS cells. Moreover, the human serum ispreferably utilized in a concentration of less than 5%, and morepreferably in a concentration of 2%. In the presence of such low serumconcentrations, the primary human dermal fibroblasts of the presentinvention will become significantly, or completely growth arrested.Hence, in this manner, two expensive and potentially complicating stepsin the autologous ORS culture system may be removed. The twocomplicating steps include: (i) removal of high serum >5%concentrations, which reduces the overall cost of the processsignificantly and; (ii) the removal of mitomycin-C treatment, whichprovides a fully mitomycin-C-free culture system and eliminates anyconcerns regarding the total elimination of the drug from the primaryculture inserts prior to the growth of the epidermal equivalents. Inaddition, the use of reduced serum concentrations allows the alternativefeeder cell-arresting procedure (i.e., the X-ray exposure step) to beeliminated, thus saving significant time and expense in the overallprocedure.

Following expansion of the ORS cells to an appropriate density (i.e.,1×10³ to 1×10⁶ cells/cm², and preferably 5×10⁴ to 1×10⁵ cells/cm²), theyare used for preparation of epidermal equivalents. Preferably, the cellsare grown to confluence. The epidermal equivalents are prepared byseeding ORS cells at an appropriate cell density (i.e., 30×10³ to100×10³ cells/cm², and preferably 60×10³ cells/cm²) within a culturedevice which is suitable for “lifting” the cells up to the air-liquidinterface during culture. Subsequently, one to four days after seeding(preferably 3 days after seeding), the ORS cells are exposed to air(e.g., by aspiration of the medium inside the insert) and the culturesare then continued for approximately 10-20 days, and preferably for14-18 days, in such “lifted” culture condition. The medium is changedperiodically during the lifted culture; preferably every two to threedays.

The present invention also encompasses skin equivalents which includeadditional layers, and so are more complex structures than epidermalequivalents. Skin equivalents comprise differentiated ORS cells as theirepidermal part and also a layer comprising a matrix component,preferably one containing embedded dermal fibroblasts and/or other cells(i.e., an “embedding matrix”). Skin equivalents are made by placing amatrix with one or more extracellular matrix substances (e.g., fibrin,fibronectin, collagens, laminins or hyaluronan or mixtures thereof) onthe upper surface of the microporous membrane described above. Whenembedding human dermal fibroblasts, preferably autologous human dermalfibroblasts, the cells are embedded at a density of 1×10³ to 1×10⁷cells/cm³; preferably 1×10⁴ to 1×10⁵ cells/cm³; and most preferablyapproximately 5×10⁴ cells/cm³. The primary culture of ORS cells is thenseeded on top of the matrix (preferably containing embedded dermalfibroblasts and/or other cells) and organotypic culturing is performedas described above. For a detailed description of the preparation ofdermal equivalents (see e.g., Limat et al., 194 Exp. Cell Res. 218-277(1991)).

It should be noted, however, that the cells which are embedded in thematrix need not be limited exclusively to dermal fibroblasts; asepidermal, mesenchymal, neuronal and/or endothelial cells can also beutilized. The embedded cells are preferably obtained from skin tissue,are more preferably allogeneic cells, and are most preferably autologouscells.

All culture steps are performed in an appropriate medium which allowsthe proliferation of the ORS cells and their outgrowth from the hairfollicles, the medium is typically changed every 2 to 3 days. Generally,the medium utilized for all steps is the same. The medium is typicallybased on a minimal medium and contains several additional ingredients.One common ingredient is serum in a concentration of 0.5-60%. In thepreferred embodiment of the present invention, human serum is used at aconcentration of less than 5%, and most preferably at a concentration of2%. Furthermore, with the development of serum-free media, it may bepossible to omit serum in toto. Epidermal growth factor (EGF) stimulatesmigration of keratinocytes and delays their senescence which results instimulation of proliferation. Cholera toxin, hydrocortisone, insulin,adenine and triiodothyronine have an effect of stimulatingproliferation. All of these ingredients are thus useful in a medium forpreparing epidermal equivalents. Nevertheless, it may be possible toomit or replace one or another of these ingredients.

Releasate from blood components (e.g., blood platelets, monocytes orlymphocytes), may serve as a source of cell proliferating activities,and therefore may substitute serum and provide other above mentionedingredients. For certain culture periods the serum-containing mediummight possibly be replaced by a defined, serum-free medium, for example,SFM (Gibco Europe, Ettlingen). The releasate from blood components(e.g., blood platelets, monocytes or lymphocytes), especially ofhomologous or autologous origin, may serve as a source of cellproliferating activities and therefore may substitute serum and provideother above mentioned ingredients or indeed may provide additionalingredients. The blood components should be added to the culture mediumin a concentration of 0.1% to 20%, and preferably 1% to 5%, after thereleasate is brought-to the same final volume as the blood from whichthese components are obtained. These releasates contain several growthfactors that are present in serum (e.g., PDGF, ECF or TGFs). However,serum as well as releasates contain many substances, and not all arecharacterized.

Releasate from blood platelets is obtained by centrifugation ofanti-coagulated whole blood, preferably human blood, in order to pelletall cells except thrombocytes. The supernatant is centrifuged once moreto spin down the thrombocytes. The thrombocytes are suspended in anappropriate buffer, e.g. phosphate buffer and treated with thrombin inorder to release their alpha granules which contain a mixture of variousgrowth factors (e.g., PDGF, PF-4, TGF-β, EGE, β-thromboglobulin). In afurther centrifugation step all cellular material is removed. Finally,the supernatant is supplemented with buffer to the volume of theoriginal blood sample from which the components are obtained. The bloodcomponents should be added to the culture medium in a concentration of0.1% to 20%; preferably 1% to 10%; and more preferably 2 to 5%.

Similarly, releasates can be obtained from other blood cells, such asmonocytes, by breaking up the cells (e.g., by sonication, freeze-thawmethod, or the like) and purifying the growth factors (e.g., byfiltration or immunological methods).

The blood component releasates can also be used to condition the woundbed in the course of grafting the epidermal or dermal equivalents.Furthermore, the culture medium containing the releasates and used toperform the organotypic culturing step, after having been conditioned bythe cells, can be used to condition the bed of the skin defect in thecourse of grafting the epidermal or dermal equivalents.

Cultivation usually is performed in inserts with microporous membranes,which contain homologous or autologous human dermal fibroblasts (HDF),especially postmitotic HDF at their undersurface. HDF secrete factorsthat condition the medium in order to get a better growth of theepidermal equivalents. The HDF layer can be formed from between 5×10³ to1×10⁵ cells/cm², and preferably approximately 1×10⁴ to 5×10⁴ cells/cm².The HDF are preferably postmitotic, but earlier passage cells can beused if they are irradiated, treated with mitomycin-C, or otherwisetreated to inhibit their proliferation but maintain their metabolism,i.e., by reduction of serum concentration.

In one embodiment, the graft thickness for the complex dermal (“complexskin”) equivalents does not exceed 0.4 mm.

Microporous membranes are suitable as a culture substrate, because theyallow substances to diffuse from one side to the other, but work as abarrier for cells. The pore size of the membrane is not a limitation onthe present invention, but should be adequate so as to allow diffusionof proteins of up to 100,000 Daltons molecular weight, and preferably ofup to 70,000 Daltons molecular weight. The membrane should at leastallow diffusion of small hormones such as insulin, and allow passage ofproteins of up to 15,000 Daltons molecular weight. Other means than amicroporous membrane for performing the function of allowing diffusionof soluble factors to the cultured ORS cells, while preventing mixing ofthe ORS cells with the HDF would also be usable.

The microporous membranes typical in the art are usually used. However,membranes fabricated from a biodegradable material (e.g., polyhyaluronicacid or polylactic acid) can also be used. When a biodegradablemicroporous membrane is employed it is contemplated that the entireculture, including the differentiated ORS cells, the microporousmembrane and the HDF, will be transplanted into the skin defect. Thus,in this alternative embodiment, the HDF grown on the underside of themembrane need not be post-mitotic or treated to preclude proliferation.While HDF tend to be less immunogenic than keratinocytes, it ispreferable that when this embodiment is employed, the HDF be allogeneiccells, preferably autologous cells.

In one embodiment, the thickness of mesh graft can range from 30-300microns. Preferably, the mesh graft thickness ranges from 0.5-0.75 mm. Agraft of tissue (for example, dermal collagen plus fibroblasts overlaidwith keratinocytes tissue) that is too thick can result in a too rapidischemic cell death, especially for the keratinocyte layer residingabove the dermal fibroblast collagen layer. By contrast, this mesh grafttissue can “take” in wound sites.

The epidermal equivalents of the present invention may range in sizefrom approximately 6 mm to approximately 2.5 cm in diameter, with apreferred diameter of 2.5 cm. For practical reasons, the experimentsdisclosed herein were performed with epidermal equivalents ofapproximately 2.5 cm in diameter.

In one embodiment, the preferred range for epidermal equivalents is50-150 microns. In a particular embodiment, the epidermal equivalentsare very thin (thinner than is generally used in the art, for example,60 microns). It has been hypothesized that making the autologous grafttoo thick will prevent a proper blood supply from being established, sothat the epidermis will not “take” at the wound site. By contrast, theepidermal equivalents of the invention can “take” in wound sites.

In many cases, however, the skin or epidermal equivalents will have tobe delivered from the facility where they are generated to theinstitution where they are used. Therefore a system is needed to enablethe transport of the skin or epidermal equivalents, which have been keptin a condition ready for grafting. Irrespective of whether themicroporous membrane is removed from the basal cell layer beforetransport, conditions resembling those during cultivation seem to befavorable. In order to keep the skin or epidermal equivalents in contactwith medium only from the basal layer, (i.e., during cultivation),agarose in a concentration ranging from 0.1% to 5%, and preferably in aconcentration of 0.5% to 1%, or methyl cellulose, or any other gelifyingsubstance in comparable concentrations, may be used to solidify thetransport medium. The skin or epidermal equivalents will be placed withtheir basal layer down on the membrane of an insert previously embeddedon top of the solidified or gelled medium. The multiwell dish containingthese inserts is then put in a blister sealed by a tyvek cover, andshipped. The skin or epidermal equivalents are, most preferably, usedfor grafting within 24 to 48 hours of initial packaging.

To improve the stability of the epidermal equivalents, the technique ofplacing a carrier membrane on top, i.e. onto the cornified aspect, ofthe epidermal equivalents and eventually adhering to it was developed.As an adhesive, fibrin glue is preferred, however, other options,including, but not limited to: extracellular matrix components such ascollagen, fibronectin, proteoglycans (e.g., hyaluronic acid, chondroitinsulfate, and the like), or basement membrane zone components (e.g.,laminin, Matrigel™, or L-polylysine), or similar tissue glues, may alsobe utilized.

The carriers utilized in the present invention may consist of asynthetic membrane, made from at one or more of the following materials(polyester, PTFE or polyurethane); from one or more biodegradablepolymers (e.g., hyaluronic acid, polylactic acid or collagen); or asilicone or vaseline gauze dressing, or any other material suitable forwound dressing. These materials which are suitable for wound dressingallow the carrier to remain in place to immobilize the implanted dermalor epidermal equivalents for several days, rather than requiring thecarrier to be removed immediately after the dermal or epidermalequivalents are transplanted. Thus, the carrier not only enhancesstability and improves handling, but it also serves as a protective coatagainst physical damage as well as the proteolytic milieu and bacteriain the wound. Moreover, it serves for orientation of the graft (i.e.,basal side down, cornified side up).

The skin or epidermal equivalents put onto the carrier have to be keptin a condition ready for grafting. Irrespective of whether themicroporous membrane is removed from the basal cell layer for transport,conditions resembling those during cultivation seem to be favorable. Inorder to keep the skin or epidermal equivalents in contact with mediumonly from the basal layer (i.e., during cultivation), agarose in aconcentration ranging from 1% to 5%, and preferably in a concentrationof 1 to 3%; methyl cellulose; or any other gelifying substance incomparable concentrations, may be used to solidify the medium. Theepidermal equivalents together with the carrier will be placed withtheir basal layer on top of the solidified or gelled medium. The wholedevice is then sealed in an air tight manner, and shipped. The epidermalequivalents are, most preferably, used for grafting within 24 hours ofinitial packaging.

The skin or epidermal equivalents are transplanted by simply placingthem in the bed of the wound or other skin defect. Preferably the skinor epidermal equivalents are then immobilized (patients are immobilizedfor 2 hours). The preferred method for immobilization is by use of abiodegradable material, by some sort of tissue glue or adequate bandage.As previously described, the bed of the skin defect can be treated withblood releasates or the medium from the organotypic culturing prior to,or concomitantly with, the transplantation.

In work using encapsulated cells devices (100 micron membrane, 200-250microns to the center of the hollow fiber), good survival of humandermal fibroblasts has been obtained at 300 micron distances from thenearest blood vessel.

Example 1 Preparation of ORS Cells

Keratinocyte precursor cells from the outer root sheath (ORS) of thehair follicles are selected and subsequently cultured by use of thefollowing methodology, as disclosed in the present invention.

Approximately 40 hair follicles were plucked with tweezers from theoccipital scalp of individuals, and those in the anagen phase, asdetected, for example, by well-developed root sheaths, were thenselected under the dissecting microscope (see e.g. Limat & Noser, 87 J.Invest. Dermatol. 485-488 (1986); Limat et al., 92 J. Invest. Dermatol.758-762 (1989)). The anagen hair was placed directly on the microporousculture insert without performing the previously-utilizedmicro-dissection to remove the hair bulbs and the infundibular hairshaft.

Generally, six anagenic hairs were explanted on the microporous membraneof a cell culture insert (Costar) that carried on its undersurface apreformed feeder layer preferably comprised of 20×10³ postmitotic humandermal fibroblasts (HDF) per cm². (see e.g., Limat et al., 92 J. Invest.Dermatol. 758-762 (1989)). The HDFs were derived from skin explants of ahealthy, repeatedly HIV-serology negative and hepatitis-serologynegative individuals and cultured in DMEM supplemented with 10% fetalcalf serum (FCS), or preferably less than 5% human serum, or mostpreferably 2% human serum.

For the purpose of obtaining an efficient outgrowth of the outer rootsheath (ORS) cells from the anagen hair and a high proliferation rate,it is important not to place the HDF feeder cells at the bottom of theculture dish, resulting in an additional medium layer between the HDFlayer and the microporous membrane supporting the ORS cells. Growingeach cell type at one side of the microporous membrane allows a veryclose interaction, but prevents cross contamination of the ORS cellswith fibroblasts and thus guarantees a pure culture of ORS cells.

The culture medium which was utilized consisted of Dulbecco's modifiedEagle's medium/F12 (3:1 v/v) supplemented with 2% human serum, 10 ng ofepidermal growth factor per ml of culture medium, 0.4 microgram ofhydrocortisone per ml, 0.135 mM adenine, and 2 nM triiodothyronine (allobtained from Sigma Chemical Co., St. Louis, Mo.). The preferred finalCa²⁺ concentration of the culture medium is 1.5 mM (see e.g., Wu et al.,31 Cell 693-703 (1982); Limat & Noser. 87 J. Invest Dermatol. 485-488(1986)). Within about 2 weeks, the ORS cells had expanded and reachedconfluence. They were dissociated with 0.1% trypsin/0.02% EDTA mixture,checked for viability, and used for preparation of epidermalequivalents. It should be noted that, although initial cultures had beenperformed using 10% fetal calf serum (FCS; Boehringer Mannheim,Germany), current utilization of human serum, in order to reduce thenumber of allogeneic ingredients, provided superior outgrowth andproliferation of the ORS cells. The human serum is preferably utilizedin a concentration of less than 5%, and most preferably in aconcentration of 2%.

Explanting plucked anagen hairs directly on the membrane of cultureinserts carrying postmitotic HDF on the undersurface as feeder cellsproved to be a simple, efficient, and reproducible method forestablishing primary cultures of ORS cells. Approximately 80% of theexplanted hair follicles gave rise to outgrowth of ORS cells, even whenderived from individuals aged more than 90 years. After 14 days, largeareas of the insert were covered by compactly arranged small cells, atwhich time they were used for preparation of epidermal equivalents ofthe present invention.

The comparison of the growth behavior of 70 strains of ORS cells, whichwere derived from a total of 30 donors, demonstrated no significantdifferences between the young (i.e., 21 donors aged 19-50 years) and theold (i.e., 9 donors aged 51-93 years) donors. Approximately 5×10⁵ cellswere generally obtained per explanted follicle and the overall degree ofcell viability was typically higher than 95%. In contrast, in theabsence of postmitotic HDF as a feeder layer, there was only sporadicoutgrowth of ORS cells from the explanted follicles.

Example 2 Preparation of Epidermal Equivalents

ORS cells harvested from primary cultures were seeded at a density of30×10³ cells/cm² to 100×10³ cells/cm², and preferably 60×10³ cells/cm²,on cell culture inserts (Costar) which had been previously inoculatedwith 10×10³ cells/cm² to 50×10³ cells/cm², and preferably 20×10³cells/cm², of postmitotic HDF cells on the undersurface of theirmicroporous membrane. Similar to the culture of ORS cells, it isimportant to keep the HDF feeder cells in close proximity with the ORScells, while concomitantly keeping them separated by use of themicroporous membrane. This culture technique enhances proliferation,differentiation, and thus the homeostasis of the developing tissue.

Culture medium was identical that that utilized for the preparation ofthe primary cultures as described supra. After 72 hours, the ORS cellswere exposed to air by aspiration of the liquid medium inside the insert(i.e., leaving the underside of the insert in contact with medium) andcultured for an additional 12-14 days, with three medium changes perweek. Alternatively, after one week lifted culture serum may be totallyomitted.

For transplantation, the so-far-utilized protocol, which is generallyemployed for preparation of the fully differentiated epidermalequivalent for wound grafting, requires the physician to carefully cutthe entire perimeter of the culture insert with a scalpel blade so as tofacilitate the release of the insert membrane (with undercoated humandermal fibroblasts) with the attached skin patch squamous-side up. Theskin patch is then released from this membrane by peeling with a fineforceps and placed, basal-side up, on a new membrane disk in a culturedish for eventual transplant to the patient. This aforementionedprocedure is both laborious and time consuming, and can lead to reversalof the basal and squamous orientation.

A markedly simpler method which utilizes a carrier membrane patch caphas been devised which utilizes a membrane patch cap (analogous to thefibrin glue patch procedure described below) which is placed directly ontop of the squamous surface layer. The membrane cap can then be easilygrasped together with the skin patch below using fine forceps and peeledfrom the culture insert well surface, and, e.g. after incubation indiluted Dispase solution, be peeled from the culture insert membrane.The membrane can then serve a plate for placing the graft onto the woundwithout mixing up the orientation of the graft (i.e., basal side down,squamous side up).

For stabilization and as a protective coating in case of grafting, theepidermal equivalents of the present invention are coated on top withdiluted fibrin glue, which also serves to clearly identify the upper(i.e., cornified) side. Fibrin glue, the preferred embodiment of thepresent invention, is a generally accepted, natural human product whichis used extensively as a tissue glue. By applying a thin coating offibrin glue (which is clearly visible with the naked eye) to thecornified squamous air-exposed surface of the epidermal equivalent, thephysician placing the epidermal equivalent onto the wound site will befully assured of proper graft orientation (i.e., the basal surface ofthe skin patch will always be the side that does not have the clearlyvisible fibrin glue cap). Previously, in many instances, during thepreparation of the epidermal patch for wound grafting, the orientationof the patch becomes confused. Should the skin patch be placed insquamous-side down orientation onto the graft site, there would besignificantly decreased likelihood of a successful graft. Thus, the useof this simple “marking” completely eliminates this problem.

In addition, anti-microbial and/or anti-fungal substances may also beincluded in the fibrin glue, so as to impede any possible microbialcontamination or overgrowth of the graft. Many chronic lesions arechronically-infected, which can result in the inhibition of graft “take”and subsequent wound healing following the initial skin grafting.Moreover, the addition of one or more antibiotics or anti-fungal agentsby direct emulsification within the fibrin glue surface cap, may providea significant improvement in the delivery of sufficient quantities ofanti-microbial agents to the transplant site.

It should be noted that the ORS cells which were harvested from primarycultures, and cultured at the air-liquid interface on insert membranescarrying postmitotic HDF at their undersurface, typically developed astratified epithelium within 14 days. This stratified epitheliumconsisted of a basal layer of small cuboidal cells below a thicksuprabasal compartment of progressively flattened cells. A prominentgranular layer, as well as an orthokeratotic horny layer were also foundto be present.

Based upon the experimental finding of approximately 80% of thefollicles giving rise to ORS cell outgrowth, approximately five anagenhair follicles were required for the generation of 1 cm² of epidermalequivalents. The period to generate graftable epidermal equivalentsusually was four weeks in toto (i.e., two weeks for the primary cultureand two weeks for the subsequent organotypic culture).

Example 3 Stabilization

Before delivery, the epidermal equivalents are “coated on-top” byplacing a silicone membrane of an appropriate diameter onto thecornified upper aspect of the cultures. To further enhance stability,e.g., in case of thin and/or large epidermal equivalents, as well as toincrease adhesion of the silicone membrane, a thin layer of tissue glue,e.g. fibrin glue, may be applied before.

On-top coating (1) enhances stability and improves handling of thegrafts, and (2) serves as a protective coat against physical damage aswell as the proteolytic milieu and bacteria in the wound.

Example 4 Shipping

On-top coated epidermal equivalents are detached from the culture insertmembranes by incubation in diluted Dispase and then grasping theepidermal equivalents together with the silicone membrane using finetweezers and transferring them on the membrane of an insert previouslyembedded in 0.7% agarose soaked with culture medium in the well of amultiwell dish. These dishes are then placed in the shipping container.For application to the wound bed, the epidermal equivalents are againgrasped, together with the silicone membrane, which (1) serves fororientation of the graft (i.e., basal side down, cornified side up) and(2) by leaving it on the grafted epidermal equivalents in the woundserves as a protective coat (see above).

Example 5 Successful Treatment of Chronic Leg Ulcers with EpidermalEquivalents Generated from Cultured Autologous Outer Root Sheath Cells

The outer root sheath cells of hair follicles can substitute forinterfollicular epidermal keratinocytes, as during healing of skinwounds when these cells migrate onto the denuded area and contribute toepidermal regeneration (Limat et al, 107(1) J. Invest. Dermatol. 128-35(1996), incorporated by reference). Using the improved culturetechniques of the invention, we generated epidermal equivalents fromcultured outer root sheath cells of patients suffering from recalcitrantchronic leg ulcers, primarily of vascular origin. In such epidermalequivalents, tissue organization as well as immunolocalization ofepidermal differentiation products (keratin 10, involucrin, filaggrin)and integrins were indistinguishable from normal epidermis. Asdetermined by the number of bromodeoxyuridine-incorporating cells, thebasal layer contained a large compartment of proliferative cellsirrespective of donor age. FACS analysis of the outer root sheath cells,used to prepare the epidermal equivalents, disclosed a fraction of smallcells with enhanced expression of β1-integrin, a potential stem cellmarker. in contrast to acute wounds, a major definitive take of graftedcultured autologous keratinocytes has not been convincingly demonstratedin chronic wounds. Grafting of epidermal equivalents generated in vitrofrom autologous outer root sheath cells on 11 ulcers in five patientsresulted in a definitive take rate of about 80%, with subsequentcomplete healing within 2 to 3 weeks of five out of seven ulcers graftedwith densely arranged cultures. This improvement in the treatment ofchronic leg ulcers with cultured autologous keratinocytes probablydepends on the large compartment of proliferative cells as well as on awell-developed horny layer which prevents disintegration of the grafts.Practical advantages of the new technique are its noninvasiveness, thelack of need for surgical facilities or anesthesia, and a shortimmobilization period after grafting.

In Vitro Experiments. Cell Cultures. About 40 hair follicles wereplucked from the occipital scalp of individuals aged up to 91 years, andthose in the anagen phase selected under the dissecting microscope. Thehair bulbs as well as the infundibular parts were removed withmicrosurgical blades. Usually, six follicles were explanted on themicroporous membrane of a cell culture insert (Falcon 3090; BectonDickinson, Franklin Lanes, N.J.) that carried on its undersurface aperformed feeder layer made of 105 postmitotic human dermal fibroblasts.Culture medium consisted of Dulbecco's modified Eagle's medium/12 (3:1)supplemented with 10% fetal calf serum (Boehringer Mannheim, German), 10ng of epidermal growth factor per ml, 0.4 μg of hydrocortisone per ml,0.1 nM choleratoxin, 0.135 mM adenine, and 2 μM triiodothyronine (allfrom Sigma Chemical Co., St. Louis, Mo.), final Ca²⁺ concentration 1.5mM. Within about 2 wk, the ORS cells expanded and reached confluence.They were dissociated with trypsin/EDTA 0.1%/0.02%, checked forviability, and grown either in secondary cultures in keratinocyte growthmedium (KGM containing 0.15 mM Ca²⁺; PromoCell, Heidelberg, Germany) orused for flow cytometry analysis and preparation of epidermalequivalents (see, below). For long-term storage in liquid nitrogen, theywere frozen in KGM containing 10% fetal calf serum and 10%dimethylsulfoxide.

For comparison, primary cultures of ORS cells were also established bytrypsinization of hair follicles and plating the disaggregated ORS cellson a preformed feeder layer made of postmitotic fibroblasts, aspreviously described (Limat et al, 1989). To avoid confusion, folliclesobtained by this method are referred to as “trypsin-treated follicles.”

Fibroblasts were derived from skin explants of a healthy, HIV-serology,and hepatitis-serology-negative individual and cultured in Dulbecco'smodified Eagle's medium supplemented with 10% fetal calf serum.

Flour Cytometry. The following mouse monoclonal antibodies (mAbs) ofIgG₁ subtype reacting with different integrin chains were used: 4B4 withthe β₁-chain (Coulter, Hialeah, Fla.), 5E8 with the α₂-chain, J143 withthe α₃-chain, Lv 230 with the α_(v)-chain, and MT78 with the α₆-chain.MAb 439-9B recognizes the β₄-chain.

ORS cells at 1×10⁶/ml were washed once with phosphate-buffered saline,1% fetal calf serum, and 0.02% NaN₃ at 4° C. and reconstituted with 1 mlof the same buffer. A 100 μl cell suspension was then incubated with 0.1μg of mAbs or isotype control antibody (Dako, Glostrup, Denmark) for 25min at 4° C. After being washed twice with the same buffer, cells wereincubated with a phycoerythrin-labeled polyclonal goat anti-mouseanti-body (Dako) for another 25 min at 4° C., washed again, andsubsequently fixed with the above-mentioned buffer supplemented with 2%paraformaldehyde. Cells were analyzed on a 4-logarithmic scale EPICSProfile II flow cytometer equipped with a power pack, and data wereanalyzed using the ELITE software (Coulter).

Epidermal Equivalents. ORS cells harvested from primary cultures wereseeded at a density of 5×10⁵/cm² on cell culture inserts (Falcon 3095)carrying 5×10⁴ postmitotic fibroblasts on the undersurface of theirmicroporous membrane. Culture medium was the same as for the preparationof primary cultures. After 24 hr. the ORS cells were exposed to air byaspiration of the medium inside the insert and then cultured for 12 to14 days with three medium changes per week. In some cultures, 65 μM5-bromo-2′-deoxyuridine (BrdU; Sigma) were added for the final 18 hr.

For histologic analysis, the epidermal equivalents were excised from theinsert with a 6 mm punch (Stiefel Laboratorium, Offenbach am Main,Germany), fixed in 5% formalin, and processed further together with theunderlying insert membrane according to standard procedures. Forimmunohistologic examination, the epidermal equivalents were similarlypunched out, but then separated from the insert membrane by finetweezers, snap-frozen in liquid nitrogen-cooled isopentane, and storedat −80° C. until processing.

For indirect immunofluorescence, cryostat sections of 6 μm wereair-dried, fixed with ice-cooled acetone/ethanol (1:1), rehydrated withphosphate-buffered saline, blocked for 15 min with nonimmune serum, andincubated at room temperature for 60 min with the primary antibodiesand, after extensive washing, for 45 min with the secondary antibodies.The following mAbs were used as primary antibodies: Ks 8.60, mainlyreacting with keratin (K) 10 and weakly with K1, diluted 1:20 (Sigma);anti-human involucrin, diluted 1:100 (Sigma); anti-humanprofilaggrin/filaggrin, diluted 1:100 (BTI, Stoughton, Mass.); 4B4directed against the β₁-integrin chain, diluted 1:10 (Coulter).Secondary mAbs against mouse IgG conjugated with fluoresceinisothiocyanate were purchased from Sigma. As negative controls, sectionswere incubated with non-immune serum and conjugated secondaryantibodies, which revealed in a few cases weak diffuse staining of fullykeratinized areas.

For the determination of BrdU-positive cells, cryostat sections weredenatured in 1.5 M HCl and successively incubated with 0.5 μg/ml Hoechst33258 for 30 min, mAb anti-BrdU (Partec, Arlesheim, Switzerland) diluted1:100 for 45 min, and fluorescein isothiocyanate-linked anti-mouse IgG(Sigma) diluted 1:30 for 45 min. The percentage of BrdU-positive cellsin the basal layer was determined in epidermal equivalents prepared fromORS cells of two leg ulcer patients aged 72 and 91 years (n=4; twoepidermal equivalents per patient). For each epidermal equivalent, about2500 basally located nuclei in 10 randomly selected sections werecounted.

For transplantation, the epidermal equivalents were excised from theinsert together with the underlying membrane using a 6-mm punch (StiefelLaboratorium) and positioned upside-down on a punched-out polyestermembrane (Thomapor 95877; Reichelt Chemie, Heidelberg, Germany) of 6 mmdiameter. In one patient, additional epidermal equivalents of 8 mmdiameter were prepared likewise. The insert membrane together with theattached postmitotic fibroblasts was then carefully removed with finetweezers. The epidermal equivalents on their supporting polyestermembrane were washed in Dulbecco's phosphate-buffered saline and leftfloating therein until their application on the wound bed, usually forno longer than 30 min.

Autologous Grafting in Chronic Leg Ulcers. With the approval of theEthics Committee of the University of Berne and after obtaining writteninformed consent, five in-patient (one male, four females, aged 58 to91) suffering from recalcitrant chronic leg ulcers (four of them withmore than two ulcers on the same leg, duration at least 4 years; venousor mixed arterial and venous disease in four. in one additional diabetesmellitus, primary lymphoedema in one) were enrolled in a pilot study.The ulcers were cleaned conventionally (primarily with hydrocolloidaldressings and topical antimicrobial agents) until ready for grafting.Then up to 20 autologous epidermal equivalents, usually 6 mm, in oneulcer 8 nm in width, were placed, basal layer downward on the surface ofthe ulcers, and the supporting polyester membranes were carefullyremoved with fine tweezers. This grafting procedure was performed at thebedside; no anesthesia was needed. In four of the patients, furtherulcers on the same leg served as controls. All ulcers were then coveredwith a transparent, semiocclusive dressing (Tegaderm; 3M, London,Canada) overlaid by an elastic bandage with compression adapted to thepatient's arterial status. The patients were immobilized for 2 himmediately after grafting. After 3 d, the semiocclusive dressing wascarefully removed and a hydropolymer dressing (Tielle: Johnson & JohnsonMedical, Ascot, UK) applied, again overlaid by the elastic bandage. Thehydropolymer dressings were then changed every 2 to 5 days. Aftercomplete re-epithelialization local treatment was switched to topicalemollients, and the patients were instructed to adhere to a long-termcompression therapy adapted to their arterial status. Take of the graftsand healing of the ulcers was documented by standardized photographstaken on each change of the dressings.

In Vitro ORS Cells Differentiate Into Epidermal Equivalents Similar toNormal Epidermis. Explanting plucked anagen hair follicles directly onthe membrane of culture inserts carrying postmitotic fibroblasts asfeeder cells at their undersurface proved to be a simple, efficient, andreproducible tool for establishing primary cultures of ORS cells. About80% of the explanted hair follicles gave rise to outgrowth of ORS cells,even when derived from individuals aged up to 91 years. After 14 days,large areas of the insert were covered by compactly arranged smallcells, at which time they were used for the preparation of the epidermalequivalents. In contrast, ORS cells derived from the trypsin-treatedfollicles exhibited a less compact arrangement with numerous cells of alarger size. Comparison of the growth behavior of 70 stains of ORS cellsderived from 30 donors revealed no significant differences between young(21 donors aged 19 through 50 years) and old donors (9 donors aged 51through 93 years), since about 0.5×10⁶ cells were usually obtained perexplanted follicle. Cell viability was higher than 95%. In the absenceof postmitotic fibroblasts, there were only sporadic outgrowth of ORScells.

Because a logarithmic linear relationship between the relative level ofβ₁-integrin on the cell surface and the proliferative capacity ofkeratinocytes has been postulated, we compared the expression ofintegrins in primary cultures of ORS cells established by the twodifferent techniques, i.e., ORS cells from explanted follicles or fromtrypsin-treated follicles. ORS cells from four different donors grown byboth techniques were analyzed by flow cytometry. On the basis of theirlight-scattering characteristics, the cells could be subdivided into twogroups; group A, with a distinctly lower forward light scatter, i.e.,smaller cell size, and group B, with higher forward light scatter, thushaving a larger cell size. For ORS cells derived from explantedfollicles, group A accounted for about 4% and group B for 72% of thetotal cell number, while values of 2.6% and 75%, respectively, werefound for ORS cells grown from trypsin-treated follicles (mean values offour separate experiments). In group A, the percentage of cells stainingfor β₁-β₄-integrins as well as the mean fluorescence per cell of the β₁-and to a lesser extent also the α₂-, α₃-, α_(v)-integrins, were higherin ORS cells grown from explanted follicles than in those fromtrypsin-treated follicles. In group B, no differences were detected inthe two culture techniques, neither in the percentage ofintegrin-positive cells nor in the mean fluorescence per cell.

ORS cells harvested from primary cultures and plated on insert membranescarrying postmitotic fibroblasts at their undersurface developed astratified epithelium within 14 days. This consisted of a basal layer ofsmall cuboidal cells below a thick suprabasal compartment ofprogressively flattened cells. A granular layer and a mostlyorthokeratotic horny layer were present.

The immunolocalization of epidermal differentiation products wasidentical to that found in normal epidermis. Thus, thedifferentiation-specific K10 was absent in the basal layer, but stronglyexpressed suprabasally from the second layer on. Involucrin displayedits typical honey-comb pattern form the mid-straturn spinosum on,whereas the granular staining of filaggrin formed a continuous bandbeneath the horny layer. As in normal epidermis, the reactivity of theα₂-, α₃- and β₁-chains of integrins was distributed over all aspects ofthe plasma membrane of the basal cells, displaying decreasing intensitywith progressive differentiation.

BrdU-positive cells were found predominantly in the basal layer of theepidermal equivalents and accounted for 24% of the basal cells [597±21BrdU-positive cells for 2464±115 basal cells (mean±SD): n=4].

Based on 80% of follicles giving rise to ORS cell outgrowth, about fiveanagen hair follicles were needed to generate 1 cm² of epidermalequivalents. The period to generate graftable epidermal equivalentsusually was 4 weeks i.e., 2 weeks for the primary culture and 2 weeksfor the organotypic culture.

Autologous Epidermal Equivalents Are Grafted Successfully on Chronic LegUlcers. A total of 11 ulcers were treated, seven of them by coveringabout 90% of the ulcer surface with densely arranged cultures, four byputting isolated cultures into the central parts. On the first change ofthe dressing 3 d after grafting, about 80% of the grafts were visibleand adherent to the wound bed in both types of treatment. Within thefollowing 2 to 3 wk the grafts consolidated in five of the seven denselygrafted ulcers, resulting in complete re-epithelialization and healing.In the two remaining, chronically infected (Pseudomonas) ulcers, thegrafts were partly destroyed, which led to delayed healing by 4 to 5weeks. In the ulcers treated by isolated grafts, there was acceleratedformation of granulation tissue and re-epithelialization mainly from thewound edges, as compared to the ulcers on the same leg treated with thedressings only. In this type of treatment, permanent take withsubsequent expansion of the grafts resulting in completere-epithelialization was only documented for one ulcer treated withlarger epithelial sheets measuring 8 mm in diameter. The control ulcersin the four patients with more than two ulcers on the same leg were onlyslightly improved after 3 weeks, at which time they were treated eitherby further grafting of autologous epidermal equivalents or byconventional surgery.

After re-epithelialization, the epidermis was initially still fragilewith some tendency to blistering after minor frictional trauma,occasionally resulting in small erosions. These erosionsre-epithelialized rapidly under conventional topical treatment. Thefirst patients have now been followed up for 6 mo and show increasingstabilization of the treated areas and no recurrence of the ulcers sofar.

From the foregoing detailed description of the specific embodiments ofthe present invention, it should be readily apparent that a uniquemethodology for the selection and culture of keratinocytes from theouter root sheath (ORS) of hair follicles for subsequent use in, forexample, skin grafting procedures, has been described. Althoughparticular embodiments have been disclosed herein in detail, this hasbeen done by way of example for purposes of illustration only, and isnot intended to be limiting with respect to the scope of the appendedclaims which follow. In particular, it is contemplated by the inventorthat various substitutions, alterations, and modifications may be madeto the invention without departing from the spirit and scope of theinvention as defined by the claims. For example, the selection of anagenhairs are believed to be a matter of routine for a person of ordinaryskill in the art with knowledge of the embodiments described herein.

1-71. (canceled)
 72. A method for treating a skin defect comprisingapplying an epidermal skin equivalent comprising keratinocyte precursorcells to the skin defect.
 73. A kit comprising an epidermal equivalent.74. A feeder cell system for producing keratinocyte precursor cellscomprising a hair follicle, feeder cells, and a microporous insertmembrane.